Project Summary Sleep regulation plays a critical role in proper neurological development. During sleep, there is enhanced synaptic plasticity in the nervous system in addition to often extensive neural remodeling. Although we know that sleep regulation is important to neurological development, many aspects of sleep regulation are poorly understood. In particular, the mechanisms of how we compensate for missed sleep in future sleep cycles are mysterious, despite general knowledge of how circadian rhythms are established. It is likely that these mechanisms involve neuropeptide signaling in the brain. However, current tools to study the roles these peptides play in sleep homeostasis, even with the use of simple model organisms like C. elegans, are low- throughput, qualitative, and subjective. In order to study sleep-like behavior in C. elegans, there is an unmet need for a tool for continuously monitoring and perturbing long-term behavior of the worm, as well as an automated and un-biased method for quantifying behavioral data. In this proposal, we will first develop the enabling technology, and use it to identify the roles of neuropeptides in homeostasis of the nematode?s sleep- like state. In Aim I, we will engineer and validate a scalable microscopy platform for monitoring and perturbing C. elegans behavior, decreasing the cost of long-term candidate screens by ~ 100 fold. In Aim 2, we will quantify behavior of wild-type animals via an automated and bias-free quantitative analysis to establish baseline behavior and prioritize behaviors that are linked to sleep homeostasis. In Aim 3, we will use the technology developed in Aims I and 2 to quantify differences in behavior of C. elegans neuropeptide mutants using a candidate approach. Because of the strong preliminary data we have gathered so far and the experience of the sponsor?s laboratory, this proposed work is likely to identify the roles of these candidate neuropeptides in sleep homeostasis, contributing to our mechanistic understanding of sleep regulation. Furthermore, the tools we develop for this particular study will also have broad utilities in many other problems, such as developmental programming, aging, and neurobehavioral studies.